Recently, attention has been given to enzymes due to their reaction specificities, substrate specificities and stereo specificities. Enzymes have been widely utilized as catalysts for medical analysis, food analysis, and the like. Among them, LAP, which is an enzyme present in blood serum, is known to be closely related to pancreas cancer, transfer of malignant tumors to the liver, diseases in the hepatic duct, etc. Accordingly, assay for LAP activity is an important inspection item in a clinical test. In assaying for the LAP activity, a method using leucine dehydrogenase has recently been utilized. The leucine dehydrogenase has been found useful due to enzymes' excellent specificities as mentioned above.
A method of assay for a trace amount using an enzyme generally involves the foregoing advantages. However, the enzyme is generally very unstable so that its catalytic activity is lost at room temperature within from several days to several weeks. This unstability is thus a serious bar with respect to assaying for a trace amount of a substance using an enzyme. Known leucine dehydrogenase is also unstable; for example, leucine dehydrogenase obtained from Bacillus sphaericus as described in J. Biol. Chem., vol. 253, page 5719 (1978) generally loses its activity in an aqueous solution (room temperature) within 1 to 3 weeks. Thus leucine dehydrogenase encounters serious disadvantages in lacking heat stability and stability over long periods of time. To best exhibit the advantages of assaying using leucine dehydrogenase it has been strongly desired to develop leucine dehydrogenase having stability to heat without losing its activity at room temperature over long periods of time.
On the other hand, in assaying for the LAP activity conventionally applied, a method which comprises using L-leucyl-.beta.-naphthylamide as a substrate and colorimetrically determining the formed .beta.-naphthylamine with p-dimethylaminobenzaldehyde, a method which comprises using L-leucyl-p-nitroanilide as a substrate and colorimetrically determining the formed p-nitroaniline, etc. are known. However, these methods are unsatisfactory in terms of specificity, etc. and were not suited for accurate measurement.
In recent years, assay methods utilizing the specificity of enzymes have also been widely adopted in the field of clinical tests. With respect to eliminating the foregoing disadvantages, these so called enzymatic methods are extremely effective. An enzymatic method has also been proposed with respect to the assay of LAP activity as shown in Japanese Patent Publication 20840/81. This method comprises reacting leucine dehydrogenase with L-leucine formed from a substrate of LAP and either measuring the amount of change of NAD.sup.+ present to NADH at an absorbancy of 340 nm, or, conjugating with a dye forming reaction and measuring with an absorbancy at the visible regions. However, this method is not practically used. That is, in this method, enzyme derived from the aforesaid Bacillus cereus, Bacillus subtilis or Bacillus sphaericus as sources is employed as leucine dehydrogenase and for this reason, it is possible to measure the LAP activity. However, there are problems in that data obtained are liable to be scattered, reproduction is poor, stability during storage is poor, enzyme per se is obtained only with difficulty and thus extremely expensive, etc. These problems become a bar to practical use.